1. Field of the Invention
When a procedure is devised for determining the presence of a sample constituent--be the devised procedure gravimetric, volumetric, spectrophotometric or whatever mode--its efficacy in producing reliable results must somehow be assessed. Otherwise, the data developed is meaningless. Hence, devising such a procedure extends far beyond building a machine, formulating reagents or developing a technique. It also must of necessity include evaluating experimental error. There must be a way of predicting the dependability of the data produced by the procedure.
The easiest, most direct way to study parameters such as reproducibility, sensitivity, accuracy and need for calibration is to subject the procedure to a test sample wherein the analyte presence and/or concentration is known beforehand, i.e., a control solution. The data furnished by the procedure can then be compared with known data and any discrepancies properly noted.
The present invention concerns itself with assessing procedures for determining the presence and/or concentration of ketone bodies in a liquid sample. Moreover, it relates to a device for preparing a ketone control solution for use in assessing the performance of various ketone body determination procedures.
2. Description of the Prior Art
Acetoacetic acid (acetylacetic acid) is a normal end product of fatty acid oxidation in the liver. It is also produced to a very limited extent by oxidative breakdown of leucine, phenylalanine, and tyrosine. .beta.-hydroxybutyric acid is formed from acetoacetic acid by reversible reduction. Acetone is produced through nonreversible decarboxylation of acetoacetic acid. ##STR2## These three substances are commonly referred to as ketone bodies or acetone bodies.
Abnormally high amounts of ketone bodies in urine or blood are referred to as ketonuria and ketonemia, respectively. These conditions can occur as a result of such diverse pathological conditions as diabetes mellitus and starvation. Because of this disease/sympton relationship, especially with diabetes mellitus, there is keen interest in the determination of ketone bodies in urine. In the case of a diabetic, the detection of ketonuria is of great significance, since a change in insulin dosage or other disease management is often indicated. Thus, strong emphasis has been placed by the medical profession on ketone body analysis, resulting in the development of a plurality of procedures sensitive to the presence and/or concentration of ketone bodies in urine.
But interest in monitoring the presence of ketones is by no means restricted to the medical profession. These compounds find a myriad of industrial applications--they are used as solvents in nitrocellulose coatings and vinyl films, they find applications in paint removers, cleaning fluids, organic synthesis, explosives manufacture, and as food additives--and in each there is a need at one time or another to perform an analytical procedure to determine ketone presence and/or concentration. These and numerous other concerns with respect to ketone presence have engendered many ketone tests.
One such test takes advantage of the propensity of ketones to react with sodium nitroprusside to give intense colors. Thus, acetone when treated with nitroprusside produces an intense red-yellow color which changes to pink-violet on acidification with acetic acid [Fritz Feigl, Spot Tests in Organic Analysis, 7th ed. (1966)]. This phenomenon occurs as a result of a coupling reaction through the NO group of the nitroprusside and the ketone to yield an isonitrosoketone which remains in the reaction mixture as a complex, colored anion. The iron (III) of the nitroprusside is reduced to its divalent state (II). It has been found that ketones which do not contain methyl or methylene groups bound to carbonyl groups are not reactive, or at least they do not produce colorforms, with nitroprusside.
The same or similar chemistry can be found in the ketone-sensitive portion of analytical reagent strips known as N-MULTISTIX.RTM. and KETO-DIASTIX.RTM. and in the reagent tablet, ACETEST.RTM., all of which are marketed by the Ames Division of Miles Laboratories, Inc. All three of these devices for determining ketone bodies are based on the nitroprusside-ketone complexing phenomenon. Thus, when the reagent strips are immersed in an aqueous ketone solution, or when the tablet is contacted with such a solution, the formation of a colored complex indicates the presence of a ketone. Moreover, the concentration of ketone can be estimated based on the intensity and hue of the color formed.
These and other methods for ketone body estimation require, as stated supra, a way of estimating their accuracy, as well as the competency of the person performing the test. One such approach is the use of a reference sample or control--a test sample in which the chemical composition and physical characteristics simulate the test samples to be analyzed. Hence, a control can be a urine sample which has been kept in the frozen state, or perhaps it comprises pooled urine which has been concentrated through freeze drying, later to be diluted to a predetermined volume.
Exemplary of a commercially available control is TEK-CHEK.RTM., marketed by the Ames Division of Miles Laboratories, Inc., which utilizes the effect of a certain pH indicator in the presence of the buffering substance used in commercially available reagent strips having ketone-responsive reagent areas. Using this ketone substitute, TEK-CHEK produces a control solution which yields a positive test for ketones with the following Ames Division products: BILI-LABSTIX.RTM., LABSTIX.RTM., KETO-DIASTIX.RTM., KETOSTIX.RTM., MULTISTIX.RTM., N-MULTISTIX.RTM. and ACETEST.RTM.. TEK-CHEK is described in product literature available from the Ames Division as comprising lyophilized urine containing a chemical substance for ketones. A substitute is used because ketones are difficult to retain in their natural state. Hence, TEK-CHEK utilizes a pH indicator to simulate a urine containing pathological amounts of ketone bodies.
Other commercially available ketone control solution products are marketed by Warner-Lambert Pharmaceutical Co. and American Hospital Supply Co., both of which products are liquids and both of which employ acetone as active agent for ketone. Another product is KovaTrol available from I.C.L. Scientific of Fountain Valley, Calif., which product must be refrigerated until use.
U.S. Pat. No. 3,920,400, issued to Scheibe, et al., discloses a uric acid standard solution wherein a lithium salt of uric acid is employed as the control substance, and a complexing agent for polyvalent metals in their higher oxidation states is also present. Typical complexing agents are specified to be malonic acid, salicylic acid, oxalic acid, glutathione, cysteine, 8-oxyquinoline and ethylenediaminetetraacetic acid. The purpose of the complexing agent additive is to stabilize and prevent the decomposition of uric acid while in solution.
Still another example of a control is that disclosed in U.S. Pat. No. 3,920,580, issued to Mast and assigned to the present assignee. There is disclosed a liquid control for glucose determination in blood or serum. It comprises water, glucose and an antidiffusing agent comprising a hydrophilic polymer.
Certain salts of cholesterol hemisuccinate are described as being useful for cholesterol controls in U.S. Pat. No. 3,859,047.
U.S. patent application Ser. No. 959,693, filed Nov. 13, 1978, and assigned to the present assignee, and which issued as U.S. Pat. No. 4,193,766 on Mar. 18, 1980, is directed to a device for preparing ketone control solutions utilizing certain metal ion complexes of acetylacetone and its homologs as a substrate. See also German Offenlegungschrift No. 27 21 681, published Nov. 13, 1978. Whereas some ketone-sensitive reagents will not respond to such acetylacetonates, other, less specific ones will. It is with the latter ketone reagents that acetylacetonates find their ability as control substrates.
To summarize the state of the art prior to the present invention, numerous control solution ingredients are known. TEK-CHEK solutions provide a substitute for ketones, a known pH indicator, which reacts with the buffering substances used in various ketone-responsive chemistries normally used in ketone determinations. Other control are equally known, such as for uric acid, glucose, cholesterol, and many others. Several liquid systems are presently marketed which contain acetone. None of the prior art controls, however, makes known the concept presently disclosed and claimed. None discloses a ketone control device which utilizes the combination of a .beta.-ketoalkanoic acid ester and alkaline substance as disclosed herein.
The present invention departs from the state of the art in dramatic fashion. No longer is it necessary to employ liquid formulations containing acetone or other liquid ketone. The present invention utilizes as an active ingredient a dry, easily storable material which, when dissolved, is directly reactive with the reagents of a ketone-sensitive test. Moreover, the invention produces a .beta.-ketoalkanoic acid in situ. The invention eliminates the need for liquid reagents and/or substitutes for ketones such as pH indicators responsive to the buffer of the ketone-sensitive reagent system. Thus, the invention provides a dry device, one which is stable upon storage and easily handled, and which provides ketone control solutions of remarkably accurate concentrations, ergo reproducibility with the ketone-sensitive test procedure.